Method of manufacturing a specifically-active albumin substance from tubercle bacilli for use as a vaccine



Patented Nov. Il, i924.

ERICH TOENNIESSEN, OF ERLANGEN, GERIVIANY.

METHOD 0F MANUFACTURING A SPECIFICALLY-ACTIVE ALBMIN SUBSTANCE FROM TUBERCLE BACILLI FOR USE AS A VACCINE.

No Drawing.

To all fte/wm t may concern:

Be it known that I, ERICH ToENNInssnN, a citizen of the German Republic, and a resident of Erlangen, Germany, have invented a new and useful Improved Method of Manufacturing a Specifically-Active Albumin Substance from Tubercle Bacilli for Use as a Vaccine, of which the following is a specification.

The vaccines produced hitherto from tubercle bacilli are delicient, although being numerous, in several respects. Fundamentally, two large groups of preparations can be distinguished, viz:

Firstly. The old tuberculines (products from the assimilation of foods by the tubercle bacilli). They are not of an albuminifen ous nature, nor by themselves able to produce anti-bodies. In cases of sickness accompanied by fever the old-tuberculine is often times therapeutically not applicable because of its toxic edect. In diagnostics it causes, when applied in a comparatively large dose, often times dangerous heart reactions, while itfails when the dose has been but small. Besides, the dosing of the oldtuberculine cannot be accurate as it cannot be separated from the alimenting bottom and cannot, therefore, be weighed.

Secondly. The bacilli bodies. In view of the lacking antreffect of the old-tuberculine it has been endeavoured to use the bacilli bodies themselves, but these latter, although having been prepared in various manners (so-called opening methods), are possessed of the drawback that they are dissolved in a mammalia organism either not at all or only with diificulty, in consequence whereof the specific albumin substances from the interior of the bacteria cell canrender their effect only in an insufhcient degree.

In view of this endeavours have been made to extract the albumin substances from the bacilli bodies by various extracting means, such, for instance, as distilled water, diluted alkalies, and acids-mention may be made also of the methods proposed by v. Ruck (Zentralblatt fr Bakteriologie; ref. vol. 58, page 244) and by Hoifmann (Wien. Klin. W. 1898)-and to apply them in diluted State to the human body. There were, however, obtained substances which were efficacious either only slightly or not Application led November 24, 1923. Serial No. 676,861.

at all, and that method has, therefore, been abandoned.

Thorough experiments have disclosed the fact that it is possible to change the difiicultly permeable wax-coverings of the bacilli by means of a special preliminary treatment of the latter, prior to the extracting process, in such a manner, that an albumin substance can now be extracted out of the interior of the bacilli bodies by means of diluted alkali, that albumin substance having never heretofore been obtained, and rendering a very strong effect in the bodies of tuberculous men.

lVhile the albumin bodies of the tubercle bacilli as obtainedby means of the known methods and applied in a dose of from 4; to 10 milligrams cause, with a tuberculous man, only slight skin extractions and only a moderate increase of temperature (as do, for instance, the albumin bodies obtained after v. Ruoks method), the albumin body obtained according to the present improved method causes very intense reactions already when a dose of from -116 milligram has been dispensed.

It appears from this that this albumin body has not been discovered by the known methods and processes, viz by extracting the bacilli by means of various dissolvents, without a preliminary treatment of the bacilli, and that the present improved method constitutes, therefore, a. fundamentally new invention. culine the new vaccine presents the advantage of forming a uniform and chemically pure substance which allows of being weighed and, thus, can be accurately dosed. Chemically it differs from the old-tuberculine by the feature that it is an albumin substance (12,6% of nitrogen, positive albumin reactions) and does not contain the specific toxic substance of the oldtubercu line (phosphorus and purinbase containing acid). It is free from phosphorus and purinbases. In spite thereof it renders a very energetic eifect'even in minute quantities.V

It is hereby proved that the tubercle bacillus contains, besides the old-tuberculine, a substance (briefly termed tuberculoprotein) having a strong speciiic ei'fect even in such minute quantities as fractions of a Over ythe oldvtubeil milligram, but being chemically fundamentally different therefrom. According to the present state of physiological chemistry the tuberculoprotein pertains to the group of the nucleines as, besides having the properties already stated, it can be precipitated by acetic acid. From/'the bacilli bodies the tuberculoprotein is distinguished essentially and very advantageously by the feature that it is separated from out of the bacilli bodies and incorporated into a genuine solution. Owing to the tuberculoprotein being in dissolved state, the amount injected 1.('contrarily to the non-resorptive bacilli-bodies) is fully yutilized and renders the etfect intended.

The .new method consists, firstly, in freeing the tubercle bacilli fromall yproduc-ts of the assimilation Vof the food (old-tuberculine), as well as from the component substances of the alimentin-g bottom, .and then treating it with idiluted muri-atie acid, wher by the ynucleine acid (themost act-ine substance of the old-tuberculline) is separated from the n-u'cl-eo-proteides land at the same time the extractiveness of the calbumin body which is free from nucleine acid .is promoted. This albumin body is extracted from the bacilli bodies with the laid of alkali, separated and .l recipita-ted by acetic acid, purified by again issolving it in alkali, precipitating it by acetic acid, and drying it ywith the acid of alcohol and ether. The tuberculoproteinthus lmanufactured and obtained in pure state can be preserved in dry condition for any period of time and permits accurate dosing as it canbe weighed.

On the base of the experiments madewith the new substance in ques-tion for theftreatment of men it can be stated that it is of reliable diagnostic and therapeutic effect. The new method is distinguished from one of the `known ones especially also by the features that with the latter method the tubercle bacilli are extracted without any preliminary treatment by means of a mixture of strong sodium ly-e and sodium hypochlorite (the known Uhlenhuts antiformine) and that the vaccine is then separated from, and precipitated out of, this solution, whereas in the present improved method the-bacilli are heated with diluted muriatic acid prior to the extraction, whereby the wax-covering is rendered permeable. Preferably the bacilliare now separated from the acid and extracted only now with the diluted alkali. After that preliminary treatment only a very diluted sodium lye is required for the extraction which contributes greatly to preserving the useful properties of the prod-uct.

lVith the limproved method one succeeds already solely by the extraction with diluted alkali in obtaining the still active substance as a uniform one, whereas the known method mentioned in the preceding paragraph requires a successive extraction by strong :1lkali, acid, and alcohol, so that a mixture of different substances is obtained.

Example: The bacilli of well-grown virulent cultures of the tubercle bacillus are separated from the alimenting liquid by filtering, washed repeatedly with 72% alcohol (by means of `a centrifugal machine), and then dried with the aid of 96% and absolute alcohol, and ether, preferably at a tempera-ture not exceeding 40o. The l' gram of dry tubercle bacillus mass is suspended uniformly by rubbing Ain gram of 5 per cent muriatic acid. The suspension is heated for 5 minutes on boiling water, then instantly cooled down and rendered moderately alkaline with 3() lper 4cent sodium lye. The liquid obtained is then again repeatedly acidulated with concentratedV muriatic acid, and ,subjected to 'centrifugal action'. The sediment is extracted for 16e-2li hours in a l per cent potash lye, the mixture stirred repeatedly during that time. The extract is filtered through clay-substances and the clear extract is moderately acidulated with acetic acid, whereafterf-ll Volume ofi() per cent acetic acid is added. The specificsubstance precipitates in liaky state, is again washed inl water, dissolved another time, with the faid of a centrifugal machine, in a l0 per cent potash lye, 'and again precipitated by acetic acid,A washed another time with water, and dried by alcohol and ether, the resulting product being a d-ry, preservable powder.

l claim: p

l. The method of producing a specifically active albumin substance from tubercle bacilli for use as a vaccine, consistingin heating the bacilli in diluted muriatic acid, separating them therefrom, extracting them in any alkali solution, and acidulating the extract with acetic acid so as to separate the albumin body therefrom. f l

2. The method of producing a specifically active albumin substance from tubercle bacilli for use as a Vaccine, consisting in freeing tubercle bacilli from their alimenting liquid, heating them in diluted muriat-ic acid, separating them therefrom, extracting them in an alkali solution, and acidulating the extract with acetic acid so as to separate the albumin body therefrom.

3. rl`he method of producing a specifically active albumin substance from tubercle bacilli for use as va vaccine, consisting in heating the bacilli in diluted mu-riatic acid, separating them therefrom, extracting them in an alkali solution, and acidulating the extract with acetic acid so as to separate the albumin body therefrom, and drying this latterby alcohol and ether. 1

4. The method of producing a specifically active albumi-n substance from tubercle bacilli for use as a vaccine, consisting in freeing tubercle bacilli from their alimenting liquid, heating them in diluted muriatic acid, Separating them therefrom, extracting them in a diluted alkali solution, acidulatng 5 the extract with acetic acid so aS to separate the albumin body therefrom, and drying this latter by alcohol and ether.

In testimony whereof I afHX my signature 1n presence of two witnesses.

ERICH TOEUNIESSEN.

Witnesses PAUL DREY, ELSLE BAER. 

